D301树脂固定化假丝酵母脂肪酶

本研究选择7种吸附和离子交换树脂进行了假丝酵母脂肪酶(Candida sp.lipase)的固定化试验,通过测定固定化后各脂肪酶的酶活,筛选出固定化效果较好的弱碱性阴离子交换树脂D301;并通过扫描电镜将D301与脂肪酶Novozym 435的表面形貌做比较,进一步选定D301树脂作为载体,并对其采用戊二醛交联固定化,研究并优化了其固定化条件。结果表明,5%戊二醛溶液的加入量为8mL,处理时间为5h,酶液浓度为1.0g/L,磷酸缓冲盐溶液pH6.0,固定化处理10h效果最好,获得的固定化酶活力可达35U/mg,酶的固定化效率约为3.5U/(mg·h)。     

固定化脂肪酶催化聚乙二醇脂肪酸酯的合成

固定化假丝酵母（Ｃａｎｄｉｄａｓｐ．）－１６１９脂肪酶催化脂肪酸和不同聚合度的乙二醇形成酯。其中以聚乙二醇４００（ＰＥＧ４００）的酯化率较高,Ｃ_１０－Ｃ_１８的饱和脂肪酸与ＰＥＧ４００反应的酯化率相仿。反应体系中,月桂酸与ＰＥＧ４００的摩尔比大于２∶１时有利于酯化反应,作用的最适温度为４０℃,最适Ｐｈ为６.０。在比较的３０种添加有机溶剂中,饱和烷烃,芳香烃能促进反应,使酯化率提高２０％左右。反应开始添加５μｌ水有利于酶的作用,外加水量超过适量时,随外加水量的增加酯化率下降。在由２.５ｍｍｏｌ月桂酸,１.２５ｍｍｏｌＰＥＧ４００,１０ｍｇ固定化脂肪酶（１００ｕ）,５ｍｌ己烷,５μｌ水组成的反应体系中,４０℃振荡反应２２ｈ,酯化率达４９.８％。经薄层色谱,气相色谱鉴定,产物为聚乙二醇４００月桂酸酯。     

氨基载体共价结合固定化海洋假丝酵母脂肪酶

共价结合法是重要的工业酶固定化方法,利用稳定的共价键固定化工业酶,在载体和酶间形成多点共价连接,可以制备稳定性较好的固定化酶,更具有实际应用价值。利用氨基载体共价结合固定化海洋假丝酵母脂肪酶,采用较为廉价的戊二醛进行辅助交联,通过单因素和正交试验,确定最佳固定化条件为:25℃、pH5. 0、0. 1%戊二醛、0. 25g载体、交联0. 5h、固定化1h、加酶量为800U,最终得到的固定化酶酶活达到83. 01U/g。固定化脂肪酶的最适pH较游离酶向碱性方向偏移,最适反应温度提高10℃,固定化酶的热稳定性和酸碱稳定性比游离酶好且重复使用性和储存稳定性明显优于游离酶。同时发现交联剂是制备固定化脂肪酶的重要因素,因此探索新型交联剂对于固定化效果的提高具有重要意义,为海洋假丝酵母脂肪酶的固定化工艺技术和工业应用奠定了良好基础。     

脂肪酶的固定化及其性质研究

采用吸附与交联相结合的方法国定化脂肪酶,研究了脂肪酶固定化的工艺条件,并考察了固定化脂肪酶的催化性能和稳定性。试验结果表明,WA20树脂固定化脂肪酶的最适条件是：酶液pH7．0、给酶量300IU／g树脂、固定时间8h,所得固定化脂肪酶的活力约为165IU／g树脂;固定化酶稳定性较高,在冰箱内贮存6个月活力没有下降,操作半衰期约为750h,而未用戌二醛文联的固定化脂肪酶操作半衰期仅约290h;固定化脂肪酶催化橄榄油水解的最适条件是：PH8．0、温度55℃、底物浓度60％（V／V）、搅拌转速500r／m。     

聚氨酯泡沫固定产脂肪酶粗状假丝酵母（Candidav alida T2）细胞的研究

对聚氨酯泡沫固定产脂肪酶粗状假丝酵母（Candida validaT2）细胞的固定化条件进行了研究。实验结果表明,聚氨酯泡沫颗粒经密度和粒径筛选,酸碱处理,以及固液比优化,载体固定细胞干质量比达到157lmg／g,细胞脂肪酶的酶活为每克干细胞1415U。电镜图片显示粗状假丝酵母菌（Candida validaT2）在载体孔隙内和脊壁上缠绕充盈,生长良好,固定结构稳定。固定化细胞连续催化水解桐籽油5批次,细胞相对水解酶活保持率达73％,固定细胞的损失率为12．5％。固定化细胞颗粒显示出良好的操作稳定性和酶活保持率,为进一步的应用研究提供了实验基础。     

聚氨酯泡沫固定产脂肪酶粗状假丝酵母(Candida valida T2)细胞的研究

对聚氨酯泡沫固定产脂肪酶粗状假丝酵母(Candida valida T2)细胞的固定化条件进行了研究.实验结果表明,聚氨酯泡沫颗粒经密度和粒径筛选,酸碱处理,以及固液比优化,载体固定细胞干质量比达到1571 mg/g,细胞脂肪酶的酶活为每克干细胞1415 U.电镜图片显示粗状假丝酵母菌(Candida valida T2)在载体孔隙内和脊壁上缠绕充盈,生长良好,固定结构稳定.固定化细胞连续催化水解桐籽油5批次,细胞相对水解酶活保持率达73%,固定细胞的损失率为12.5%.固定化细胞颗粒显示出良好的操作稳定性和酶活保持率,为进一步的应用研究提供了实验基础.     

酵母表面展示脂肪酶合成己二酸二异辛酯

展示酶的酵母细胞既具有固定化酶的优点,又有制备简单、成本较低的特点.采用表面展示南极假丝酵母脂肪酶B (Candida antarctica lipase B,CALB)的毕赤酵母细胞催化合成己二酸二异辛酯(Diisooctyl adipate,DIOA),对该反应体系进行优化,并实现了初步工艺放大制备.经条件优化后,在10mL反应体系中,DIOA的产率可达85.0％.该工艺放大到200mL反应体系时,DIOA产率可达97.8％.经减压蒸馏,DIOA纯度可达到98.2％.该酵母表面展示脂肪酶在合成绿色润滑油己二酸二异辛酯中具有良好应用前景.     

改性壳聚糖固定化脂肪酶的研究

以化学改性后的壳聚糖为载体固定假丝酵母99-125脂肪酶,研究了不同的活化剂对壳聚糖表面羟基基团的活化程度,及以活化后壳聚糖为载体采用不同固定化方法对假丝酵母脂肪酶固定效果的影响。结果表明1-乙基-3-(3-甲基氨基)丙基碳二亚胺可有效的活化壳聚糖表面羟基,活化后的壳聚糖表面氨基与戊二醛偶联后形成的壳聚糖为良好的脂肪酶固定化载体,其固定脂肪酶的水解活力可高达86.8U/g。此外,还对影响固定化进程中的各种因素进行了研究,确定最优条件,比较了固定化前后酶的热稳定性、有机溶剂稳定性及最适反应温度。并考察了该固定化脂肪酶催化合成棕榈酸十六酯的操作稳定性,结果表明,连续反应16批之后棕榈酸十六酯的转化率仍能达到85%以上。     

毕赤酵母表面展示南极假丝酵母脂肪酶B全细胞催化合成葡萄糖月桂酸酯

利用表面展示南极假丝酵母脂肪酶B(Candida antarctica lipase B,CALB)的毕赤酵母细胞为全细胞催化剂,以葡萄糖为酰基受体,月桂酸为酰基供体,在非水相体系中催化合成糖酯。用硅胶柱层析对产物进行初提,再用制备液相色谱进一步分离纯化,并用高效液相色谱-质谱鉴定纯品性质。对该酶法合成糖脂反应体系进行了优化,其中考察了有机溶剂种类、复合溶剂体系中二甲基亚砜(DMSO)体积百分比、酶量、底物摩尔比、水活度和温度等几个影响酯化反应的因素。结果表明:在5mL反应体系中,以叔戊醇/二甲基亚砜(DMSO30%,V/V)为反应介质,添加初始水活度为0.11的全细胞催化剂0.5g,葡萄糖0.5mmol/L,月桂酸1.0mmol/L,60°C下反应72h后,葡萄糖月桂酸单酯的转化率达到48.7%。     

Studies of the Heterogeneity of a Candida cylindracea (rugosa) Lipase: Monitoring of Esterolytic Activity and Enantioselectivity by Chiral Liquid Chromatography

A method for the determination of lipase activity in terms of rate and enantioselectivity of hydrolysis of a chiral ester substrate has been developed. When this method was applied to fractions, isolated from preparative, column chromatographic separations (anion-exchange, molecular sieve) of the lipase, significant differences in enantioselectivity (E) was found between the fractions. The highest enantioselectivity was found in the first main peak obtained on DEAE-Sepharose chromatography, meaning that the enzyme with the highest isoelectric point shows the highest esterolytic enantioselectivity.

The experimental results are discussed in the light of some earlier reported results and with respect to the possible existence of subunit aggregates and isoenzymes.     

Enhanced Resolution of a Secondary Alcohol by Hydrolysis of a Bichiral Ester Catalyzed by Lipase from Candida cylindracea

The effect of a chiral centre in the acyl group on the resolution of esters prepared from a racemic alcohol was investigated. R-2-chloropropionic acid afforded a higher enantiomeric ratio than S-2-chioropropionic acid in the hydrolysis of the corresponding esters of racemic 1-phenylethanol catalyzed by Candida cylindracea lipase. Even when a mixture of esters prepared from racemic acid and racemic alcohol was used for resolution of the alcohol, a noteworthy high enantioselectivity was observed. The hydrolysis of a bichiral ester offers an amplification in the resolution of enantiomers of alcohols by the combination of a chemical diastereoselectivity and an enzymatic enantio- and diastereoselectivity.     

Enhanced Resolution of a Secondary Alcohol by Hydrolysis of a Bichiral Ester Catalyzed by Lipase from Candida cylindracea

The effect of a chiral centre in the acyl group on the resolution of esters prepared from a racemic alcohol was investigated. R-2-chloropropionic acid afforded a higher enantiomeric ratio than S-2-chioropropionic acid in the hydrolysis of the corresponding esters of racemic 1-phenylethanol catalyzed by Candida cylindracea lipase. Even when a mixture of esters prepared from racemic acid and racemic alcohol was used for resolution of the alcohol, a noteworthy high enantioselectivity was observed. The hydrolysis of a bichiral ester offers an amplification in the resolution of enantiomers of alcohols by the combination of a chemical diastereoselectivity and an enzymatic enantio- and diastereoselectivity.     

Lipase kinetics: hydrolysis of triacetin by lipase from Candida cylindracea in a hollow-fiber membrane reactor

The aptitude of a hollow-fiber membrane reactor to determine lipase kinetics was investigated using the hydrolysis of triacetin catalyzed by lipase from Canadida cylindracea as a model system. The binding of the lipase to the membrane appears not to be very specific (surface adsorption), and probably its conformation is hardly altered by immobilization, resulting in an activity comparable to that of the enzyme in its native form. The reaction kinetics defined on the membrane surface area were found to obey Michaelis-Menten kinetics. The specific activity of the lipase in the membrane reactor was found to be significantly higher than in an emulsion reactor. The activity and stability of the enzyme immobilized on a hydrophilic membrane surface seem not to be influenced significantly by the choice of the membrane material. The hollow-fiber membrane reactor is a suitable tool to assess lipase kinetics in a fast and convenient way.     

Alcohol Induced Reversal of Enantioselectivity in a Lipase Catalyzed Resolution of 2-Chloropropionic Acid

Alcohol induced reversal of enantioselectivity in the esterification of 2-chloropropionic acid using lipase from Candida cylindracea has been investigated. It was found that an alcohol having substituents both at the α- and the β-carbon preferentially esterified the S-acid, while a straight chain alcohol preferentially esterified the R-acid. Intermediate enantioselectivities were obtained with alcohols having substituents either at the α- or the β-carbon, but still in favor of the R-acid. An acyl binding domain composed of three subsites is proposed for this lipase; one for the hydrocarbon chain, a second for a methyl substituent and a third for an electronegative substituent.     

蚕丝固定化脂肪酶的研究

研究了蚕丝固定化脂肪酶的工艺条件,并考察了固定化脂肪酶的稳定性。试验结果表明：蚕丝与对-β-硫酸酯乙砜基苯胺(SESA)进行反应的最适条件是PH=10．8,SESA：2．0g／g蚕丝,反应生成的对氨基苯磺酰乙基蚕丝(ABSE-蚕丝)经重氮化后与脂肪酶偶联的最适条件是：pH=7．5,偶联时间>10h。加酶量为168～308u／g蚕丝时,所得固定化脂肪酶活力为106～160u√g蚕丝．此时固定化冀的活力回收率较高(>52％)。固定化脂肪酶稳定性较高．其操作半衰期约为250h。     

Lipase activity in vesicular systems: characterization of candida cylindracea lipase and its activity in polymerizable dialkylammonium surfactant vesicles

Lipase from Candida cylindracea (CCL) was incorporated into polymerizable positively charged dialkylammonium bromide surfactant vesicles. The enzyme was incorporated by the use of the dehydration-rehydration method or by incubation. In the latter case, trapping efficiencies of up to 100% could be obtained. Activities of free and vesicle-incorporated CCL were tested for three triglycerides: triacetin, tributyrin, and tricaprylin. Enzyme activity was lowest in homogeneous mixtures (triacetin and small concentrations of tributyrin) and highest in heterogeneous mixtures (tricaprylin and high concentrations of tributyrin). Entrapment in vesicular systems is advantageous, especially in homogeneous reaction mixtures and in the case of the production of insoluble fatty acid (caproate), because inhibition by the acid can be suppressed. The influence of several surface-active additives, including vesicles, on the activity of lipase in triglyceride assays was tested. Vesicles have a positive influence on the activity, whereas other positively charged additives act as inhibitors. In the case of tricaprylin assays, the positively charged additives increase the activity. Finally, tryptic digestion for free and incorporated CCL were compared. Free CCL is readily inactivated, whereas incorporated enzyme is protected from proteolytic degradation. (c) 1993 John Wiley & Sons, Inc.     

非水相脂肪酶催化有机硅醇的酯化反应

非水相酶催化是酶工程研究热点之一。本文介绍了来自Ｃ．ｃｙｌｉｎｄｒａｃｅａ的脂肪酶催化有机硅醇和脂肪酸的酯化反应。该酶可催化有机硅醇与脂肪酸的酯化反应,并对不同链长的脂肪酸底物、有机溶剂极性及水含量等进行了初步研究。     

脂肪酶产生菌分离,鉴定及酶性质的研究

从含油污泥中分离筛选出１７株产脂肪酶菌株,对其中一株进行鉴定,为无花果丝孢酵母（Ｔｒｉｃｈｏｓｐｆｉｇｕｅｒｉａｅ）．研究了该菌的最适产酶条件,并对其部分酶性质进行了研究．